AP2
Clathrin adaptor
4x adaptin subunits
PICK1 binds alpha appendage
Clathrin binds beta appendage
Competitively competes with NSF for binding to GluA2
GST Pulldown
To see whether 2 proteins can interact (in a test-tube)
Can use to identify binding site
Fusion protein plasmid - GST + protein 1
Plasmid taken up by bacteria - expresses the fusion protein
Bacteria are good at making GST proteins, therefore good at making fusion proteins
Bind GST fusion protein to glutathione-coated agarose beads
Incubate with protein of interest (2)
Centrifuge
SDS-Page + Western blot - AB protein of interest (2)
2nd AB w/ chemiluminescence enzyme
Expose to photographic film to detect light - dark band = protein of interest
Ensure ABs are validated
Good for testing optimal conditions for a reaction to occur
ie. Buffer - with/without ATP, b-SNAP etc.
Co-immunoprecipitation
To see if 2 proteins interact in native tissue
Protein of interest bound to AB on sepharose bead covered in protein A/G
Incubate in brain lysate
Centrifuge
SDS-Page + Western blotting
Immunocytochemistry co-localisation
To see if 2 proteins are in the same place within a cell
Fix with formaldehyde (in order to access IC proteins)
Primary AB for protein 1 w/ fluorescence dye
Primary AB for protein 2 w/ fluorescence dye
Confocal microscopy - overlap, different colour = co-localising
Confocal = limited to the wavelength of light ~180 nm
Transfection
- Virus ie. Sindbus, lentivirus = engineer to encode virus; encourage viral DNA to incorporate into host cell’s DNA
- Lipofection = liposome associates with DNA encoding protein of interest; forms a DNA-lipid complex; lipid fuses with PM, releases DNA
- Biolistics = DNA-coated gold bullets
Control - gold w/ no DNA; ensure gold is inert
Show that LTD is mediated by clathrin-mediated endocytosis
Disrupt amphiphysin-dynamin
Amphiphysin associates with the neck of the clathrin-coated pit, recruits dynamin (GTPase) via SH3 domains which pinches off the newly formed vesicle
Memory requiring LTD
Novel object recognition memory = perihinal cortex
Blocked w/ in vivo expression of AP2-GluA2 peptide
Memory requiring LTP
Spatial object memory = hippocampus
Forgetting = GluA2-internalisation
-promoted natural forgetting with peptide: GluA2-NSF
-prevent long-term natural forgetting with peptide: GluA2-AP2 (no endocytosis)
Promote endocytosis
Peptide to disrupt: GluA2-NSF
NSF stabilises AMPARs at the synapse
In vivo memory tasks
Disrupt GluA2-AP2
- memory disrupted in the perihinal cortex (novel recognition memory)
- speed up natural forgetting process in the hippocampus (spatial memory -object location memory)
Disrupt GluA2-NSF (stabilise AMPARs at the synapse)
-prevent natural long-term memory loss
Forgetting in the hippocampus occurs due to GluA2-containing AMPARs being internalised!
The persistence of learning depends on the maintenance of steady-state level of synaptic GluA2-containing AMPARs = requires interaction with NSF-GluA2
Disrupt GluA2-NSF
GluA2-NSF interaction discovered via yeast 2-hybrid scanning
Promote endocytosis (NSF cannot stabilise AMPARs at the synapse)
Occlude LTD - therefore involved in mechanism of LTD!
Disrupt GluA2-AP5
Inhibit GluA2-endocytosis
Endosomal markers
Early endosome = EEA1
Recycling endosomes = Rab11, Syntaxin13
Late endosomes = LAMP1
Which interactions compete for the same binding site?
GluA2-NSF (stabilise AMPARs at synapse)
GluA2-AP2 (involved in clathrin-mediated endocytosis)
What amino acids do kinases require to recognise a phosphorylation site?
Example!
Lysine residue
ie. LTD = phosphorylation @ Ser880 on GluA2
Block P from occuring my mutating lysine resiue (K–>A) = reduced LTD
Need phosphorylation at Ser880 for FULL LTD to occur!
Phosphorylation of Ser880
P @ Ser880 on GluA2 occurs during LTD
By PKC:
For = phorbol esters increase phosphospecific AB staining
Against = PKC inhibition (ie. CalC/BIS) does not stop P-Ser880 or LTD from occuring
Could be - multiple kinases act to P-Ser880!
Inhibit phosphorylation = reduce LTD
Capacitance studies = GluA2 is recycled faster to the synapse (PICK1 cannot bind GluA2 + retain from the synapse)
Hypothesis: PICK1 binds PKC
-interaction brings PKC close to GluA2 to P-Ser880 - PICK1 acts as a scaffold for PKC
BUT - PICK1 K/O - P-Ser880 still occurs!
Future - try inhibiting PICK1-PKC binding site (avoid problems associated with knocking-out proteins)
PICK1-GluA2 = Ca relationship
Peak ~ 15uM
Biphasic - could explain how increasing [Ca] results in either LTD or LTP
Smaller [Ca] increase = LTD
Larger [Ca] increase = LTP
***timings are also important;
LTD = prolonged, modest increase
LTP = transient, large increase
It is possible that a Ca-sensing protein will respond to specific patterns of Ca accumulation over time
Deletion of NTD (glutamate + aspartate aa) = no biphasic relationship - no LTD occurring - need Ca sensor to trigger LTD
Need PICK1 to bind Ca (+ dimerise) in order to drive internalisation of AMPARs - therefore no LTD can occur
Ca binding PICK1
Binds to region near NTD
Acidic amino acids = glutamate + aspartate
Circular dichromism studies (with/without Ca bound) + thermal denaturation studies = decrease in intramolecular bonds + destabilisation of tertiary interactions
Expose BAR domains = can dimerise
DIFFERENT TO EXO = proteins involved contain C2 domains!
BAR domains
Highly conserved dimerisation domains - involved in membrane dynamics
Contains +vely charged lysine residues - mediate binding to the -vely charged phospholipid heads
ie. PICK1, amphiphysin
GluA2-NSF
Interaction stabilises GluA2-containing AMPARs at the surface
NSF binds to a juxtamembrane region of GluA2
Co-factor = alpha-SNAP (binds to PICK1’s BAR domain)
SNAPs act as a physical connection with NSF and PICK1!
The mechanical force of ATP hydrolysis can be transferred, via SNAPs, to dissociate PICK1-GluA2 = ‘rotational shearing’ effect
It is unclear whether PICK1 dimers dissociate into monomers = maybe try FRET experiments???
GluA2-NSF interaction
- Enhanced via nitrolyslation!!!
- Ca-sensitive: basal Ca favours binding; high [Ca] blocks interaction (LTD) - allows PICK1-mediated endo to occur
~15uM
-optimal GluA2-PICK1 binding
-inhibits GluA2-NSF
(remember NSF competes with AP2 binding to GluA2)
Inhibiting GluA2-NSF = occludes LTD (see a rundown in EPSC prior to LFS)
LTD
P @ Ser880 on GluA2 by PKC
Inhibition of GluA2-NSF
Experimentally inducing LTP
High frequency stimulation/theta burst stimulation
Glycine
Experimentally inducing LTD
Low frequency stimulation
NDMA (activate extra-synaptic receptors)
Co-factor to NSF
Alpha-SNAP
Deletion of alpha-SNAP = embryonic death
NSF+SNAP regulate PICK1-GluA2 interactions AND they regulate the SNARE complex
Beta-SNAP = Block effect - stops NSF from working
